J.L. Ortega-Vinuesa

A comparative study between the adsorption of IgY and IgG on latex particles.

The use of egg yolk antibodies (IgY) instead of IgG from mammalian species may present several advantages in the development of routine diagnostic immunoassays. On the one hand, the animal suffering is reduced, as antibodies are obtained directly from the egg. On the other hand, the use of IgY avoids the rheumatoid factor interference. The rheumatoid factor interacts with IgG molecules in many immunoassays causing false positive results. Despite these advantages, IgY antibodies are scarcely used. As part of an aim to develop a diagnostic test based on IgY-latex agglutination, a preliminary study on some characteristics of the IgY-latex complexes is carried out. In this work, protein adsorption and desorption, isoelectric point, electrokinetic mobility, and colloidal stability are analysed. Results are compared to those obtained by IgG. Interesting differences are observed (which mainly arise from the difference in molecular structure between IgY and IgG), suggesting that IgY is a more hydrophobic molecule than IgG. In addition, colloidal dispersions of IgY-covered latex particles are more stable (at pH 8) than those sensitized by IgG.

Role of wettability and nanoroughness on interactions between osteoblast and modified silicon surfaces.

Development of new biomaterials is a constant in regenerative medicine. A biomaterials surface properties, such as wettability, roughness, surface energy, surface charge, chemical functionalities and composition, are determinants of cell adhesion and subsequent tissue behavior. Thus, the main aim of this study was to analyze the correlation between changes in wettability without topographical variation and the response of osteoblast-like cells. For this purpose oxidized silicon surfaces were methylated to different degrees. Additionally, the influence of nanoroughness, and the subsequent effect of hysteresis on cell behavior, was also analyzed. In this case oxidized silicon pieces were etched with caustic solutions to produce different degrees of nanoroughness. Axisymmetric drop-shape analysis and atomic force microscopy confirmed that the proposed surface treatments increased the nanometer roughness and/or the water contact angles. MG-63 osteoblast-like cells were cultured on the altered surfaces to study proliferation, and for ultrastructural analysis and immunocytochemical characterization. Increasing the nanometer surface roughness or water contact angle enhanced osteoblast behavior in terms of cell morphology, proliferation and immunophenotype, the effect provoked by methylation being more significant than that caused by nanoroughness.

Chitosan nanocapsules: Effect of chitosan molecular weight and acetylation degree on electrokinetic behaviour and colloidal stability

In recent years, chitosan nanocapsules have shown promising results as carriers for oral drug or peptide delivery. The success in their applicability strongly depends on the stability of these colloidal systems passing through the digestive tract. In gastric fluids, clear stability comes from the high surface charge density of the chitosan shell, which is completely charged at acidic pH values. However, in the intestinal fluid (where the pH is almost neutral) the effective charge of these nanocapsules approaches zero, and the electrostatic forces cannot provide any stabilization. Despite the lack of surface charge, chitosan nanocapsules remain stable in simulated intestinal fluids. Recently, we have demonstrated that this anomalous stability (at zero charge) is owed to short-range repulsive forces that appear between hydrophilic particles when immersed in saline media. The present work examines the influence of the chitosan hydrophobicity, as well as molecular weight, in the stability of different chitosan nanocapsules. A study has been made of the size, polydispersity, electrophoretic mobility, and colloidal stability of eight core-shell nanocapsule systems, in which the chitosan-shell properties have been modified using low-molecular-weight (LMW) and high-molecular-weight (HMW) chitosan chains having different degrees of acetylation (DA). With regard to the stability mediated by repulsive hydration forces, the LMW chitosan provided the best results. In addition, contrary to initial expectations, greater stability (also mediated by hydration forces) was found in the samples formed with chitosan chains of high DA values (i.e. with less hydrophilic chitosan). Finally, a theoretical treatment was also tested to quantify the hydrophilicity of the chitosan shells.

A review of factors affecting the performances of latex agglutination tests

The present review describes the different strategies followed to improve the performance of latex agglutination tests. The analysis is mainly focused on the diverse parameters that affect the final colloidal stability of the immunoprotein-latex system. These parameters include: the surface properties of polymer carriers; the different kind of antibodies usually employed; the use of BSA as stabilizer; the co-adsorption of various macromolecules (BSA, surfactants and lipids) and antibodies; recent approaches to colloidal stability at high ionic strengths due to hydration forces; and the covalent coupling of antibodies on functionalized latex particles. Special emphasis is given to the relation between electrophoretic mobility and the colloidal stability of the sensitized particles and how this knowledge can be utilized for a better understanding of the immunoagglutination kinetic.

Stagnant versus dynamic conditions: a comparative adsorption study of blood proteins.

Haemodynamic parameters of flowing blood, such as diffusion, convection, flow and shear rates, are important as they determine the interaction of cells with vessel walls and prosthetic implants in the cardiovascular system. Most of the studies under flow conditions have been performed with platelets or other cells, and less attention has been paid to the effects that these parameters may cause on the adsorption of proteins. For this reason we studied how different shear rates affect the adsorption of human albumin, fibrinogen, total serum proteins, and complement factors 1q and 3c from human serum to silicon surfaces. The most relevant results indicate that during non-flow conditions the amount of adsorbed proteins is always lower than under flow. The different shear rates (225, 915, 1800 and 2700 s(-1)) all gave similar results, indicating that such a parameter is not very critical for single protein deposition. The differences in kinetics of complex protein solutions are conveniently highlighted by use of specific polyclonal antibodies. The difference between non-flow or low shear rate conditions and physiological flow conditions was enhanced for the complement cascade system.

Adsorption of antibody onto Pluronic F68-covered nanoparticles: link with surface properties

The use of nanoparticles as drug delivery systems is an emerging application to improve intravenous therapy. Controlling the biocompatibility of the nanoparticles is a crucial step towards the optimal implementation of these systems. Adsorption of serum components onto the nanoparticles is driven mainly by hydrophobic forces. Thus, incorporation of hydrophilic polymers such as polyethylene oxide (PEO) derivatives to the nanostructure surface reduces the interaction of nanoparticles with blood stream components (IgG). The effectiveness of the poloxamer for reducing protein adsorption depends on the resistance of this coating layer. A fundamental understanding of the properties of this surface coating is crucial towards the rational design of these systems. Here, we have used an innovative combination of experimental techniques to evaluate the properties of the nanoparticles and more specifically, the mechanical properties of the coating. Electrophoretic mobility and colloidal stability data suggest similar surface characteristics between IgG–Pluronic–polystyrene (PS) and IgG–PS complexes, indicating that the protein adsorption is just slightly reduced by the presence of poloxamer. Nevertheless, the biological activity of the adhered antibodies suggests that the Pluronic F68 significantly altered their immunoactivity. The decrease in the activity might indicate a partial denaturation of the protein and/or changes in the preferential orientation when adsorbing caused by the surfactant–protein interactions. The surface characterisation of the IgG layers adsorbed onto a Pluronic covered surface importantly provides evidence of the conformational change undergone by the protein, supporting the partial protein denaturation suggested by the loss of immunoreactivity in the IgG–Pluronic–PS particles. The use of surface tension to obtain structural and mechanical information about the coating procedure is a novel approach to understand generic features of the biocompatibility of colloidal systems. These results may help to understand why drug nanocarriers coated by poloxamers improve their long-circulating properties in comparison with uncoated particles.

Characterization of Different Functionalized Lipidic Nanocapsules as Potential Drug Carriers

Lipid nanocapsules (LNC) based on a core-shell structure consisting of an oil-filled core with a surrounding polymer layer are known to be promising vehicles for the delivery of hydrophobic drugs in the new therapeutic strategies in anti-cancer treatments. The present work has been designed as basic research about different LNC systems. We have synthesized—and physico-chemically characterized—three different LNC systems in which the core was constituted by olive oil and the shell by different phospholipids (phosphatidyl-serine or lecithin) and other biocompatible molecules such as Pluronic® F68 or chitosan. It is notable that the olive-oil-phosphatidyl-serine LCN is a novel formulation presented in this work and was designed to generate an enriched carboxylic surface. This carboxylic layer is meant to link specific antibodies, which could facilitate the specific nanocapsule uptake by cancer cells. This is why nanoparticles with phosphatidyl-serine in their shell have also been used in this work to form immuno-nanocapsules containing a polyclonal IgG against a model antigen (C-reactive protein) covalently bounded by means of a simple and reproducible carbodiimide method. An immunological study was made to verify that these IgG-LNC complexes showed the expected specific immune response. Finally, a preliminary in vitro study was performed by culturing a breast-carcinoma cell line (MCF-7) with Nile-Red-loaded LNC. We found that these cancer cells take up the fluorescent Nile- Red molecule in a process dependent on the surface properties of the nanocarriers.

Thermally sensitive reversible microgels formed by poly(N-Isopropylacrylamide) charged chains: A Hofmeister effect study

In this study, we present a new method to obtain anionic and cationic stable colloidal nanogels from PNIPAM charged chains. The stability of the particles formed by inter-chain aggregation stems from the charged chemical groups attached at the sides of PNIPAM polymer chains. The particle formation is fully reversible-that is, it is possible to change from stable polymer solutions to stable colloidal dispersions and vice versa simply by varying temperature. In addition, we also demonstrate that the polymer LCST (lower critical solution temperature), the final particle size and the electrokinetic behavior of the particles formed are highly dependent on the electrolyte nature and salt concentration. These latter results are related to Hofmeister effects. The analysis of these results provides more insights about the origin of this ionic specificity, confirming that the interaction of ions with interfaces is dominated by the chaotropic/kosmotropic character of the ions and the hydrophobic/hydrophilic character of the surface in solution.

Ion‐Specific Aggregation of Hydrophobic Particles

This work shows that colloidal stability and aggregation kinetics of hydrophobic polystyrene (PS) nanospheres are extremely sensitive to the nature of the salt used to coagulate them. Three PS latices and four aggregating electrolytes, which all share the same cation (Na(+)) but have various anions located at different positions in the classical Hofmeister series depending on their kosmotropic or chaotropic character, are used. The present study focuses on analyzing different aggregating parameters, such as critical coagulation concentrations (CCC), cluster size distributions (CSD), initial kinetic constants K(11), and fractal dimensions of the aggregates d(f). While aggregation induced by SO(4)(2-) and Cl(-) behaved according to the predictions of the classical Derjaguin-Landau-Verwey-Overbeek theory, important discrepancies are found with NO(3)(-), which become dramatic when using SCN(-). These discrepancies among the anions were far more significant when they acted as counterions rather than as co-ions. While SO(4)(2-) and Cl(-) trigger fast diffusion-limited aggregation, SCN(-) gives rise to a stationary cluster size distribution in a few aggregation times when working with cationic PS particles. Clear differences are found among all analyzed parameters (CCC, CSD, K(11), and d(f)), and the experimental findings show that particles aggregate in potential wells whose depth is controlled by the chaotropic character of the anion. This paper presents new experimental evidence that may help to understand the microscopic origin of Hofmeister effects, as the observations are consistent with appealing theoretical models developed in the last few years.

Salt Effects in the Cononsolvency of Poly(N‐isopropylacrylamide) Microgels

Poly(N-isopropylacrylamide) (PNIPAM) is well known to exhibit reentrant behavior or cononsolvency in response to the composition of a mixed solvent consisting of water and a low-chain alcohol. Since the solvent structure plays an important role in this phenomenon, the presence of structure-breaking/structure-making ions in solution is expected to have a dramatic effect on the cononsolvency of PNIPAM. The present work examines the way that the presence of different salts can modify the reentrant-phase diagram displayed by a cationic PNIPAM microgel in the mixed ethanol/water solvent. The effects of four Hofmeister anions--SO(4)(2-), Cl(-), NO(3)(-) and SCN(-)--with different abilities to modify the solvent structure are analyzed. The species with kosmotropic or structure-making character show a clear competition with ethanol for the water molecules, intensifying the nonsolvency of the PNIPAM with the EtOH volume fraction (phi(e)). However, striking results are found with the most chaotropic or structure-breaking anion, SCN(-). In contrast to what happens in water-rich solutions, the presence of SCN(-) in alcohol-rich solvents enhances the solubility of the polymer, which macroscopically results in the microgel swelling. Moreover, this ion displays great stabilizing properties when phi(e)> is 0.2. These results have been explained by considering how chaotropic or structure-breaking ions interact with water and ethanol molecules.